ABSTRACT Efficient DNA repair is a double-edged sword. Accurate repair of such deleterious DNA lesions as double- stranded breaks, inter-strand crosslinks, and damaged replication forks promotes genome stability. It also allows cancer cells to acquire a more aggressive character and develop resistance to radiation and DNA damaging chemotherapeutics. Additionally, untimely deployment and/or misregulation of the DNA repair machines may further destabilize the genome (which can lead to cancer) or may result in the accumulation of toxic repair intermediates (which can lead to cell death). Significant gaps remain in our understanding of the molecular events that funnel the intermediates of otherwise accurate repair into ?rogue?, genome- destabilizing mechanisms. This research program emphasizes the molecular machinery of homologous recombination, how it is integrated into DNA replication, repair and recombination (the 3Rs of genome stability), and how it is misappropriated in the molecular pathways that process stalled DNA replication events and DNA breaks through highly mutagenic, genome destabilizing mechanisms. Our central hypothesis is that the activities of the RAD51 recombinase, the ssDNA-binding protein RPA, recombination mediators BRCA2 (in human) and Rad52 (in yeast), and DNA repair helicases are finely tuned by a variety of factors, which include posttranslational modifications, interacting partner proteins, specific DNA structures and DNA lesions. These factors affect the protein conformational dynamics and critical protein- protein interfaces. Understanding how the protein plasticity and kinetics of assembly of the macromolecular machines of DNA repair will show us new ways to selectively manipulate the activities of RAD51 and multifunctional DNA helicases in DNA replication and repair. We are leveraging and building the tools of single-molecule biochemistry, biophysics and chemical biology. Our unique perspective on the formation, activities and regulation of the nucleoprotein complexes orchestrating recombination is rooted in our ability to sort individual human DNA repair proteins with their native posttranslational modifications, and to probe and separate activities associated with different surface- tethered proteins and nucleoprotein complexes at the single-molecule level. Our goal is to provide an entirely new outlook on how the cell balances the assembly and activities of the molecular machines that can repair, but also destabilize, the genome, and to be able to alter this balance with new anticancer chemotherapeutics.